Biomarkers for seizures

ABSTRACT

The application relates to markers for seizures and epilepsy. Polypeptide expression panels or arrays are provided, comprising one or more probes capable of binding specific polypeptides in blood plasma or blood serum of a mammalian subject. Also provided are methods for detecting seizure, methods for predicting seizure, use of sICAM-5 in the treatment of seizure, methods for assessing the effectiveness of a treatment of seizure, and diagnostic kits.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/001,306, filed Aug. 23, 2013, which is a national stage ofInternational Patent Application No. PCT/US2012/026467, filed Feb. 24,2012 (expired), which claims the benefit of the priority of U.S.Provisional Patent Application No. 61/446,461, filed Feb. 24, 2011(expired), which applications are incorporated herein by reference intheir entireties.

BACKGROUND OF THE INVENTION

Epilepsy is presently characterized by at least two unprovoked seizures,although other definitions are evolving. It is currently estimated toaffect 50 million people worldwide with 200,000 new cases diagnosed everyear in the United States alone. Current methods for diagnosing epilepsyare laborious and inaccurate. Differential diagnosis for epilepsytypically involves a neurological exam, patient history, neural imagingand electroencephalography (EEG). While EEGs are considered the mostuseful test in confirming a diagnosis of epilepsy, there are significantfalse positives from this test, and to a lesser extent, false negatives.Between 10% and 40% of people with epilepsy will have normal EEGresults, even over several tests. The costs of the EEG may also not beunderstated, both in money and in time. No tests are available todetermined imminent risk of seizure or risk of recurrence.

SUMMARY OF THE INVENTION

This application is directed toward a blood test for epilepsy diagnosis.The application provides individual and panels/arrays of biomarkersindicative of seizure or a tendency to have seizure. In one embodiment,a polypeptide expression panel or array is provided comprising a probecapable of binding soluble ICAM-5 (i.e., sICAM-5 or sICAMS) in bloodplasma or blood serum of a mammalian subject, wherein a decreased plasmaor serum concentration of sICAM-5 relative to a healthy control isindicative of seizure or a tendency to have seizure. Further panelscomprise probes capable of binding TARC and/or IL-2, IL-6, IL-8, IL-1β,IFN-γ, and in combination with sICAM-5. Still further panels compriseprobes capable of binding IL-10, IL-12 p70, Fas, Fas-ligand, MCP-1, MDC,MIP-1β, GM-CSF, MCP-4, IL-10, BDNF, Eotaxin, Eotaxin-3, and/or TNF-α,and in combination with sICAM-5 and/or TARC.

Also provided are methods for detecting seizure, methods for assessingthe effectiveness of a treatment of seizure, a tendency to have seizure,or treatment of an underlying disorder resulting in seizure, anddiagnostic kits.

Other aspects and advantages of the present invention are describedfurther in the following detailed description of the preferredembodiments thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a model for inflammation and epilepsy.

FIGS. 2A-2C are charts summarizing the assay of sICAMS in plasma fromepilepsy and control patients. FIG. 2A is a dot-plot of sICAMSconcentrations in plasma (⋄, controls; □, patients). The dotted line isthe cut-line that best discriminates between patients and controls. FIG.2B is a bar graph and error calculation for data in FIG. 2A. Differenceis significant for p=0.003. FIG. 2C is a ROC curve for data in FIG. 2A,showing an area under the curve (AUC) value of 0.803.

FIGS. 3A-3C are charts summarizing the assay of TARC in plasma fromepilepsy and control patients. FIG. 3A is a dot-plot of TARCconcentrations in plasma (⋄, controls; □, patients). The dotted line isthe cut-line that best discriminates between patients and controls. FIG.3B is a bar graph and error calculation for data in FIG. 3A. Differenceis significant for p=0.035. FIG. 3C is a ROC curve for data in FIG. 3A,showing an area under the curve (AUC) value of 0.759.

FIGS. 4A-4C are charts summarizing the assay of TARC/sICAMS ratio inplasma from epilepsy and control patients. FIG. 4A is a dot-plot ofTARC/sICAMS concentration ratio in plasma (⋄, controls; □, patients).The dotted line is the cut-line that best discriminates between patientsand controls. FIG. 4B is a bar graph and error calculation for data inFIG. 4A. Difference is 17.1 fold, and is significant for p=0.025. FIG.4C is a ROC curve for data in FIG. 4A, showing an area under the curve(AUC) value of 1.00.

FIGS. 5A-5C are charts summarizing an assay of IL-6 in plasma fromepilepsy and control patients. FIG. 5A is a dot-plot of IL-6concentrations in plasma (⋄, controls; □, patients). The dotted line isthe cut-line that best discriminates between patients and controls. FIG.5B is a bar graph and error calculation for data in FIG. 5A. Differenceis 2.8-fold, and is significant for p=0.012. FIG. 5C is a ROC curve fordata in FIG. 5A, showing an area under the curve (AUC) value of 0.821.

FIGS. 6A-6C are charts summarizing an assay of IL-6/sICAMS ratio inplasma from epilepsy and control patients. FIG. 6A is a dot-plot ofIL-6/sICAMS concentration ratio in plasma (⋄, controls; □, patients).The dotted line is the cut-line that best discriminates between patientsand controls. FIG. 6B is a bar graph and error calculation for data inFIG. 6A. Difference is 9.9 fold, and is significant for p=0.05. FIG. 6Cis a ROC curve for data in FIG. 6A, showing an area under the curve(AUC) value of 0.90.

FIGS. 7A-7C are charts summarizing an assay of IL-8 in plasma fromepilepsy and control patients. FIG. 7A is a dot-plot of IL-8concentrations in plasma (⋄, controls; □, patients). The dotted line isthe cut-line that best discriminates between patients and controls. FIG.7B is a bar graph and error calculation for data in FIG. 7A. Differenceis 1.4-fold, and significant for p=0.002. FIG. 7C is a ROC curve fordata in FIG. 7A, showing an area under the curve (AUC) value of 0.715.

FIGS. 8A-8C are charts summarizing an assay of IL-8/sICAMS ratio inplasma from epilepsy and control patients. FIG. 8A is a dot-plot ofIL-8/sICAMS concentration ratio in plasma (⋄, controls; □, patients).The dotted line is the cut-line that best discriminates between patientsand controls. FIG. 8B is a bar graph and error calculation for data inFIG. 8A. Difference is 6.5-fold, and is significant for p=0.017. FIG. 8Cis a ROC curve for data in FIG. 8A, showing an area under the curve(AUC) value of 0.88.

FIGS. 9A-9C are charts summarizing an assay of IL-113 in plasma fromepilepsy and control patients. FIG. 9A is a dot-plot of IL-1βconcentrations in plasma (⋄, controls; □, patients). The dotted line isthe cut-line that best discriminates between patients and controls. FIG.9B is a bar graph and error calculation for data in FIG. 9A. Differenceis significant for p=0.003. FIG. 9C is a ROC curve for data in FIG. 9A,showing an area under the curve (AUC) value of 0.803.

FIGS. 10A-10C are charts summarizing an assay of IL-2 in plasma fromepilepsy and control patients. FIG. 10A is a dot-plot of IL-2concentrations in plasma (⋄, controls; □, patients). The dotted line isthe cut-line that best discriminates between patients and controls. FIG.10B is a bar graph and error calculation for data in FIG. 10A.Difference is significant for p=0.003. FIG. 10C is a ROC curve for datain FIG. 10A, showing an area under the curve (AUC) value of 0.788.

FIGS. 11A-11C are charts summarizing an assay of IFN-γ in plasma fromepilepsy and control patients. FIG. 11A is a dot-plot of IFN-γconcentrations in plasma (⋄, controls; □, patients). The dotted line isthe cut-line that best discriminates between patients and controls. FIG.11B is a bar graph and error calculation for data in FIG. 11A.Difference is 2.4-fold, and significant for p=0.01. FIG. 11C is a ROCcurve for data in FIG. 11A, showing an area under the curve (AUC) valueof 0.701.

FIGS. 12A-12C are charts summarizing an assay of GM-CSF in plasma fromepilepsy and control patients. FIG. 12A is a dot-plot of GM-CSFconcentrations in plasma (⋄, controls; □, patients). The dotted line isthe cut-line that best discriminates between patients and controls. FIG.12B is a bar graph and error calculation for data in FIG. 12A.Difference is 1.2-fold, and significant for p=0.297. FIG. 12C is a ROCcurve for data in FIG. 12A, showing an area under the curve (AUC) valueof 0.554.

FIGS. 13A-13C are charts summarizing an assay of BDNF in plasma fromepilepsy and control patients. FIG. 13A is a dot-plot of BDNFconcentrations in plasma (⋄, controls; □, patients). The dotted line isthe cut-line that best discriminates between patients and controls. FIG.13B is a bar graph and error calculation for data in FIG. 13A.Difference is 1.1-fold, and significant for p=0.383. FIG. 13C is a ROCcurve for data in FIG. 13A, showing an area under the curve (AUC) valueof 0.527.

DETAILED DESCRIPTION OF THE INVENTION

In many epilepsies, an immune response is generated within the region ofseizure onset. In several distinct tissue lesion types such as tuberoussclerosis (TSC) and mesial temporal sclerosis (MTS), pro-inflammatorycytokines such as IL-1, IL-6, TNF-α, Fas, and Fas-ligand are activated.In addition, there is complement fixation and deposition, alteredblood-brain barrier permeability, and macrophage infiltration.Inflammation may generate a wide variety of downstream effects includingupregulation of IL-1β production, activation of TLR4, NFκB, mTOR, andMAPK cascades, attraction of activated lymphocytes, microglia, andmacrophages, and alteration of astrocyte physiology. Without being boundby theory, these changes may be a result of a disease process leading toseizures, caused by seizures, and/or be the result of seizures (See FIG.1). The present application addresses a need in the art for markersassociated with seizures.

As used herein, the abbreviations “A1AT” and “α1AT” refer to alpha1-antitrypsin, also known as serpin peptidase inhibitor, clade A(alpha-1 antiproteinase, antitrypsin), member 1.

The terms “comprising” and “including” are used interchangeably, unlessotherwise noted.

The term “cryptogenic” is used herein to refer to a seizure or epilepsyof unknown origin.

The terms “disease”, “disorder”, or “condition” are used herein to referto any manifestations, symptoms, or combination of manifestations orsymptoms, recognized or diagnosed as leading to, causing, or influencingseizure. The terms include, but are not limited to, traumas,inflammatory and autoimmune responses, physiological malformations, andgenetic defects.

The abbreviation “GM-CSF” refers to granulocyte-macrophagecolony-stimulating factor.

The abbreviation “HGF” refers to hepatocyte growth factor.

The abbreviation “ICAM-1” refers to intercellular adhesion molecule 1.

The term “ictal” refers to a physiologic state or event such as aseizure.

The term “indicative” (or “indicative of”) encompasses both prediction(including tendency), and detection (proximate to the occurrence of aseizure), and unless otherwise noted, embodiments encompassing the termare intended to define and encompass embodiments specific to prediction,specific to detection, and for prediction as well as for detection of apast or current event. Use of the term indicative in conjunction withthe term “tendency” is intended solely for emphasis of evidence of apast event versus a tendency toward a future event, but the use solelyof indicative is intended to encompass tendency unless otherwiseindicated.

The abbreviation “BDNF” refers to brain-derived neurotrophic factor.

The abbreviation “MCP-1” refers to monocyte chemotactic protein-1, alsoknown as chemokine (C-C motif) ligand 2 (CCL2), or variants thereof.

The abbreviation “MDC” refers to macrophage derived cytokine, also knownas C-C motif chemokine 22 (CCL-22), or variants thereof.

The abbreviation “MIP-10” refers to macrophage inflammatory protein-1β,also known as chemokine C-C motif ligand 4 (CCL-4), or variants thereof.

The abbreviation “IP-10” refers to interferon gamma-induced protein 10,small-inducible cytokine B10, C-X-C motif chemokine 10 (CXCL10), orvariants thereof.

Eotaxin, also known as eotaxin-1, refers to chemokine (C-C motif) ligand11 (CCL11), or variants thereof.

Eotaxin-3 refers to chemokine (C-C motif) ligand 26 (CCL26), or variantsthereof.

The term “sample” is used herein to refer to a blood plasma or bloodserum sample, unless otherwise noted. In each embodiment describedherein, the use of blood plasma is contemplated as an independentembodiment from the alternative of blood plasma or blood serum. In eachembodiment described herein, the use of blood serum is contemplated asan independent embodiment from the alternative of blood plasma or bloodserum. In each embodiment described herein, the use of anotherbiological sample, including but not limited to cerebrospinal fluid(CSF) and a tissue sample obtained by resection, is contemplatedaccording to conventional techniques in the art for obtaining the sampleand for analysis of same. The sample can be treated prior to use, suchas preparing plasma from blood, diluting viscous fluids, and the like.Methods of treatment can involve filtration, distillation, extraction,concentration, inactivation of interfering components, the addition ofreagents, and the like.

The terms “seizure” and “epilepsy” are used interchangeably, twounprovoked seizures being required for a clinical diagnosis of epilepsy,unless otherwise noted. The term epilepsy may also be defined by theunderstanding of, or theories of, seizure as understood as of the filingof the application.

The terms “subject”, “individual”, and “patient” are usedinterchangeably herein to refer to a mammal from which a sample istaken, unless otherwise noted. The terms are intended to encompassembodiments specific to humans. A subject, individual or patient may beafflicted with, at risk for, or suspected of having a tendency to haveseizure or a disorder for which seizure is symptomatic. The term alsoincludes domestic animals bred for food or as pets, including horses,cows, sheep, pigs, cats, dogs, and zoo animals. Typical subjects fortreatment include persons susceptible to, suffering from or that havesuffered one or more seizures. In particular, suitable subjects fortreatment in accordance with the invention are persons that aresusceptible to or that have suffered one or more seizures.

The abbreviation “TARC” refers to ‘thymus and activation regulatedchemokine’, and is used interchangeably herein with chemokine (C-Cmotif) ligand 17 (CCL17).

The terms “telencephalin”, “TLN”, “ICAM-5”, and “ICAMS” are usedinterchangeably herein.

The term “tendency”, e.g., “tendency to have seizure”, is intended torefer to a reasonable medical probability of an event, e.g., seizure tooccur or recur. The term also encompasses the frequency with which suchevents may occur before, after, or during ongoing treatment.

As used herein, the term “treat” or “treating” refers to any method usedto partially or completely alleviate, ameliorate, relieve, inhibit,prevent, delay onset of, reduce severity of and/or reduce incidence ofone or more symptoms or features of a particular condition, e.g.,seizure or a seizure-related disorder. Treatment may be administered toa subject who does not exhibit signs of a condition and/or exhibits onlyearly signs of the condition for the purpose of decreasing the risk ofdeveloping pathology associated with the condition. Thus, depending onthe state of the subject, the term in some aspects of the invention mayrefer to preventing a condition, and includes preventing the onset, orpreventing the symptoms associated with a condition. The term alsoincludes maintaining the condition and/or symptom such that thecondition and/or symptom do not progress in severity. A treatment may beeither performed in an acute or chronic way. The term also refers toreducing the severity of a condition or symptoms associated with suchcondition prior to affliction with the condition. Such prevention orreduction of the severity of a condition prior to affliction refers toadministration of a therapy to a subject that is not at the time ofadministration afflicted with the condition. Preventing also includespreventing the recurrence of a condition, frequency thereof, or of oneor more symptoms associated with such condition. The terms “treatment”and “therapeutically” refer to the act of treating, as “treating” isdefined above. The purpose of intervention is to combat the conditionand includes the administration of therapy to prevent or delay the onsetof the symptoms or complications, or alleviate the symptoms orcomplications, or eliminate the condition. For example, a treatment maybe used to ameliorate symptoms or frequency thereof (e.g., frequency ofseizure) associated with a disorder.

The terms “tuberous sclerosis”, “tuberous sclerosis complex”, and theabbreviation/acronyms “TS” and “TSC”, are used interchangeably herein.

The abbreviation “VCAM-1” refers to vascular cell adhesion molecule 1.

The abbreviation “VEGF-A” refers to vascular endothelial growth factorA.

In one embodiment, a polypeptide expression panel or array is provided,the panel or array comprising a probe capable of binding soluble ICAM-5(sICAM-5) in blood plasma or blood serum of a mammalian subject, whereina decreased plasma or serum concentration of sICAM-5 relative to ahealthy control is indicative of seizure or a tendency to have seizure.In another embodiment, a polypeptide expression panel or array isprovided, the panel or array comprising a probe capable of binding TARCin blood plasma or blood serum of a mammalian subject, wherein anincreased plasma or serum concentration of TARC relative to a healthycontrol is indicative of seizure or a tendency to have seizure.

Also provided is a polypeptide or array comprising a probe capable ofbinding sICAM-5 in blood plasma or blood serum and a probe capable ofbinding TARC in blood plasma or blood serum of a mammalian subject,wherein a decreased plasma or serum concentration of sICAM-5 incombination with an increased plasma or serum concentration of TARC(relative to a healthy control) indicates seizure or a tendency to haveseizure. In further embodiments, the increase of the ratio ofTARC/sICAM-5 in tested subjects relative to control (healthy,non-epileptic/non-seizure) is greater than 20, greater than 17, greaterthan 15, greater than 10, greater than 5, or greater than 1. The ratiomay also be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, or fractional increments thereof, e.g. 1.4 and 1.5. In otherembodiments, the ratio is 2 or more whole or fractional standarddeviations above the mean for controls.

The above panels or arrays may also include one or more probes capableof binding one or more of IL-2, IL-6, IL-8, IL-1β, and IFN-γ, wherein anincreased plasma or serum concentration of one or more relative to ahealthy control is indicative of seizure or a tendency to have seizure.

In still further embodiments, the polypeptide expression panel or arraysdescribed above may further include one or more probes capable ofbinding IL-10, IL-12 p70, Fas, Fas-ligand, MCP-1, MDC, MIP-1β, GM-CSF,MCP-4, IP-10, BDNF, Eotaxin-3, Eotaxin, and TNF-α, wherein an alteredplasma or serum concentration of one or more of IL-10, IL-12 p70, Fas,Fas-ligand, MCP-1, MDC, MIP-1β, GM-CSF, MCP-4, IP-10, BDNF, Eotaxin-3,Eotaxin, and TNF-α (relative to a healthy individual) indicates atendency to have seizure. In further embodiments, the patient is ahuman.

In another embodiment, a method for predicting or detecting seizure isprovided, comprising contacting a blood plasma or blood serum sampleobtained from a mammalian subject with a diagnostic reagent that canmeasure or detect the expression level of soluble ICAM-5 (sICAM-5) andcontacting a blood plasma or blood serum sample obtained from amammalian subject with a diagnostic reagent that can measure or detectthe expression level of TARC, wherein a decreased plasma or serumconcentration of sICAM-5 relative to a healthy control in combinationwith an increased plasma or serum concentration of TARC indicates aseizure having occurred or a tendency to have seizure.

The method may also include contacting the blood plasma or blood serumsample with one or more diagnostic reagents that can measure or detectthe expression level of IL-2, IL-6, IL-8, IL-1β, and IFN-γ, whereinaltered plasma or serum concentration of one or more of IL-2, IL-6,IL-8, IL-1β, and IFN-γ relative to a healthy control indicates atendency to have seizure. Still further the method may includecontacting the blood plasma or blood serum sample with a diagnosticreagent that can measure or detect the expression level of one or morediagnostic reagents that can measure or detect the expression level ofIL-10, IL-12 p70, Fas, Fas-ligand, MCP-1, MDC, MIP-1β, GM-CSF, MCP-4,IP-10, BDNF, Eotaxin-3, Eotaxin, and TNF-α, wherein altered plasma orserum concentration of one or more of IL-10, IL-12 p70, Fas, Fas-ligand,MCP-1, MDC, MIP-1β, GM-CSF, MCP-4, IP-10, BDNF, Eotaxin-3, Eotaxin, andTNF-α relative to a healthy control indicates a seizure having occurredor a tendency to have seizure.

In yet another embodiment, a method for assessing the effectiveness of atreatment of seizure or a disorder for which seizure is symptomatic isprovided, the method including contacting a first blood plasma or bloodserum sample obtained from a mammalian subject prior to treatment withone or more diagnostic reagents that can measure or detect theexpression level of soluble ICAM-5 (sICAM-5) and/or TARC, and contactinga second blood plasma or blood serum sample obtained from a mammaliansubject subsequent to treatment with a diagnostic reagent that canmeasure or detect the expression level of soluble ICAM-5 (sICAM-5)and/or TARC, wherein an increased plasma or serum concentration ofsICAM-5 and/or a decreased level of TARC in the second blood plasma orblood serum sample relative to the first blood plasma or blood serumsample indicates effectiveness in treatment of seizure or a disorder forwhich seizure is symptomatic. The method may further include contactingthe first blood plasma or blood serum sample and the second blood plasmaor blood serum sample with one or more diagnostic reagents that canmeasure or detect the expression level of IL-6, IL-8, IL-2, IL-1β,IFN-γ, IL-10, IL-12 p70, Fas, Fas-ligand, MCP-1, MDC, MIP-1β, GM-CSF,MCP-4, IP-10, BDNF, Eotaxin-3, Eotaxin, and TNF-α, wherein an alteredconcentration of IL-6, IL-8, IL-2, IL-1β, IFN-γ, IL-10, IL-12 p70, Fas,Fas-ligand, MCP-1, MDC, MIP-1β, GM-CSF, MCP-4, IP-10, BDNF, Eotaxin-3,Eotaxin, and TNF-α in the second blood plasma or blood serum samplerelative to the first blood plasma or blood serum sample indicateseffectiveness in treatment of seizure or a disorder for which seizure issymptomatic.

In still further embodiments, a method for determining the whether ornot one or more seizures are resultant from inflammation, comprisingcontacting a blood plasma or blood serum sample obtained from amammalian subject with a diagnostic reagent that can measure or detectthe expression level of soluble ICAM-5 (sICAM-5) and/or contacting ablood plasma or blood serum sample obtained from a mammalian subjectwith a diagnostic reagent that can measure or detect the expressionlevel of TARC, wherein a decreased plasma or serum concentration ofsICAM-5 relative to a healthy control and/or an increased plasma orserum concentration of TARC indicates an inflammatory basis or componentof seizure. The method may further include contacting the blood plasmaor blood serum sample with one or more diagnostic reagents that canmeasure or detect the expression level of IL-6, IL-8, IL-2, IL-1β,IFN-γ, IL-10, IL-12 p70, Fas, Fas-ligand, MCP-1, MDC, MIP-1β, GM-CSF,MCP-4, IP-10, BDNF, Eotaxin-3, Eotaxin, and TNF-α, wherein an alteredconcentration of IL-6, IL-8, IL-2, IL-1β, IFN-γ, IL-10, IL-12 p70, Fas,Fas-ligand, MCP-1, MDC, MIP-1β, GM-CSF, MCP-4, IP-10, BDNF, Eotaxin-3,Eotaxin, and TNF-α in the blood plasma or blood serum sample indicatesan inflammatory basis or component of seizure.

In yet other embodiments, a method for determining the whether or notseizure is likely to occur in a subject is provided, comprisingcontacting a blood plasma or blood serum sample obtained from amammalian subject with a diagnostic reagent that can measure or detectthe expression level of soluble ICAM-5 (sICAM-5) and/or contacting ablood plasma or blood serum sample obtained from a mammalian subjectwith a diagnostic reagent that can measure or detect the expressionlevel of TARC, wherein a decreased plasma or serum concentration ofsICAM-5 relative to a healthy control and/or an increased plasma orserum concentration of TARC indicates a tendency to have seizure. Themethod may further include contacting the blood plasma or blood serumsample with one or more diagnostic reagents that can measure or detectthe expression level of IL-6, IL-8, IL-2, IL-1β, IFN-γ, IL-10, IL-12p70, Fas, Fas-ligand, MCP-1, MDC, MIP-1β, GM-CSF, MCP-4, IP-10, BDNF,Eotaxin-3, Eotaxin, and TNF-α, wherein an altered concentration of IL-6,IL-8, IL-2, IL-1β, IFN-γ, IL-10, IL-12 p70, Fas, Fas-ligand, MCP-1, MDC,MIP-1β, GM-CSF, MCP-4, IP-10, BDNF, Eotaxin-3, Eotaxin, and TNF-α in theblood plasma or blood serum sample indicates a tendency to have seizure.

In specific further embodiments of the above, the seizure is associatedwith a temporal lobe epilepsy. In a further embodiment, the temporallobe epilepsy is mesial temporal sclerosis (MTS). In other embodiments,the seizure is associated with tuberous sclerosis complex (TSC).

In still other specific further embodiments of the above, the seizuremay be cryptogenic. In further embodiments, the seizure is notassociated with immune response to a pathogen.

The embodiments, including the probes and panels/arrays of probes,described herein may be used to detect whether or not a seizure has (islikely to have occurred). They may also be used to predict thelikelihood of further seizure. Additionally, they may be used to predictwhether or not seizure is likely following a brain injury or headtrauma. They are also useful in identifying whether or not a seizure isthe result of an inflammatory process. Further, they may be used inassessing whether or not a treatment is effective.

ICAM-5 is a neuron-derived protein differentially distributed in theblood plasma or blood serum of epilepsy patients relative to healthypatients. Soluble ICAM-5 (also known as sICAMS, sICAM-5, or variantsthereof) is cleaved from ICAM-5 by metalloproteases in response toinflammation. Unexpectedly, it is found that decreased sICAM-5expression is found in the case of seizure patients relative to healthypatients. As reflected in Example 1 herein, sICAM-5 expression is adiagnostic marker better than any presently available. Further, asreflected in Table 4 (see Example 3) herein, other markers are alsoindicative of a tendency to have seizure.

TARC is also an effective marker, differentially distributed in theblood plasma or blood serum of epilepsy patients relative to healthypatients and it is shown to be elevated in seizure patients. Incombination, the sICAM-5/TARC ratio is significantly elevated (ratio of17.1) over healthy control. Additional markers that are useful include,alone or in combination, IL-1β, IL-2, IL-8, and IFN-γ. Still additionalmarkers that are useful include, alone or in combination, IL-10, IL-12p70, Fas, Fas-ligand, MCP-1, MDC, MIP-1β, GM-CSF, MCP-4, IP-10, BDNF,Eotaxin-3, Eotaxin, and TNF-α. Probes may further include α1AT, VCAM-1,ICAM-1, HGF, and VEGF-A. Probes may also include those for components ofthe complement cascade, e.g., C1q, C3c and C3d.

By way of non-limiting example, the following polypeptide panels orarrays are embodiments of the application (the terms decreased,elevated, and altered refer to the expression level in the epilepticpatient versus that in a healthy subject):

-   -   sICAM-5 (decreased);    -   sICAM-5 (decreased), TARC (increased);    -   sICAM-5 (decreased), TARC (increased), IL-1β (increased), IL-6        (increased), IL-8 (increased);    -   sICAM-5 (decreased), TARC (increased), IL-1β (increased), IL-2        (increased), IL-6 (increased), IL-8 (increased), IFN-γ        (increased);    -   sICAM-5, GM-CSF, BDNF;    -   sICAM-5, GM-CSF, BDNF, IL-1β;    -   sICAM-5, IL1β, IL-6;    -   sICAM-5, BDNF, IL-12 p70;    -   GM-CSF, BDNF;    -   GM-CSF, BDNF;    -   sICAM-5, GM-CSF, IFN-γ, IL-10, IL-12 p70, IL-113, IL-2, IL-6,        IL-8, TNF-α;    -   sICAM-5, GM-CSF, BDNF, IFN-γ, IL-10, IL-12 p70, IL-1β, IL-2,        IL-6, IL-8, TNF-α;    -   GM-CSF, IFN-γ, IL-10, IL-12 p70, IL-1β, IL-2, IL-6, IL-8, TNF-α;    -   GM-CSF, BDNF, IFN-γ, IL-10, IL-12 p70, IL-1β, IL-2, IL-6, IL-8,        TNF-α; and    -   sICAM-5, TARC, IL-1β, IL-2, IL-8, IFN-γ IL-10, IL-12 p70, Fas,        Fas-ligand, MCP-1, MDC, MIP-1β, GM-CSF, MCP-4, IP-10, BDNF,        Eotaxin-3, Eotaxin, TNF-α.

Also provided is a diagnostic kit comprising a polypeptide expressionpanel or array as described herein. The kit may also be predictive,useful in determining imminent risk of seizure or recurrence of seizure,or in assessing recurrence risk. In one embodiment, the kit is for thediagnosis of a temporal lobe epilepsy, such as MTS. In anotherembodiment, the kit is for the diagnosis of tuberous sclerosis complex(TSC). The kit may also contain a syringe and/or vile for drawing blood.The kit will contain one or more probes corresponding to the polypeptidemarkers of the panel or array. The kit may also an ELISA plate. Amultiple and portable (M&P) ELISA may also be provided as part of a kitof an embodiment. Still other suitable components will be known to oneof skill in the art, and are encompassed hereby.

Samples may be obtained from patients by conventional techniques. Thesetechniques may include those covered by an institutional review board(IRB) approved protocol. In one embodiment, the samples areanticoagulated using sodium citrate. In a further embodiment, plasma isprepared by centrifuging samples, e.g., at 5,000 g (g=gravity) for 15minutes at 4° C. Controls may also be purchased from commercial vendors.

Levels (concentrations) of the polypeptide to be quantified in plasmamay be obtained by any of a number of methods known in the art, theparticular procedure not being a limitation of the embodiments herein.For example, ELISA, Indirect ELISA, Sandwich ELISA, Competitive Elisa,and Multiple and Portable (M&P) ELISA may be used. Probes specific tothe antigen (polypeptide or marker) to be detected may be obtainedcommercially or designed by techniques known in the art. In oneembodiment for sICAM-5 detection, protein G affinity purified mousemonoclonal anti-human ICAM-5 antibody is used as the capture antibody.Single- and Multi-probe kits are available from commercial suppliers,e.g., Meso Scale Discovery. These kits include the kits referenced inthe Examples hereto.

Also described herein are methods of treating or preventing seizure or adisorder for which seizure is symptomatic in a mammalian subject,comprising delivery of sICAM-5. In a further embodiment, the mammal is ahuman. Also provided is use of sICAM-5 to treat or prevent seizure or adisorder for which seizure is symptomatic in a mammalian subject, anduse in preparing a medicament therefor. Given that ICAM-5 is expressedon the surface of telencephalic neurons (i.e., is localized to thebrain), treatment or prevention may be effected without undesiredsystemic effects.

Treatment or prevention may be made intravenous or viaintra-cerebrospinal fluid (intra-CSF) by techniques known to one ofskill in the art. Delivery may also be made by any other suitable means,including by intranasal delivery to the CSF with a suitable carrier orexcipient.

EXAMPLES

The invention is now described with reference to the following examples.These examples are provided for the purpose of illustration only and theinvention should in no way be construed as being limited to theseexamples but rather should be construed to encompass any and allvariations that become evident as a result of the teaching providedherein. The specific embodiments described in the Examples are intendedto be embodiments of the invention.

Example 1 Evaluation of ICAMS, TARC, and Other Polypeptides (Alone andin Combination) as Blood Plasma Markers of Seizure or a TendencyTherefor

Sample Collection and Processing

Blood samples are collected from human epilepsy patients. The samplesare anticoagulated using Na-citrate, and the plasma is prepared bycentrifuging samples at 5,000×G for 15 minutes at 4° C. The supernatantsolutions are then aliquoted and stored at −80° C. Followingcentrifugation, the supernatant solutions are aliquoted and frozen at−80° C. Samples of plasma, also anticoagulated with Na-citrate, arepurchased from commercial vendors. Differences among sets of controlsare not significant where p>0.05.

Detection/Quantification of sICAM-5

Levels of immunoreactive Telencephalin/ICAM-5 in plasma were measured bysandwich ELISA using electrochemiluminescence detection. Assays werecarried out on high bind SECTOR® Imager 6000 reader plates (Meso ScaleDiscovery (MSD), Gaitherburg, Md.) as follows. Wells were coatedovernight with protein G affinity purified mouse monoclonal anti-humanICAM-5 antibody (capture antibody; R&D Systems, Minneapolis, Minn.;catalog # MAB 1950), 2 μg/ml diluted in phosphate buffered saline (PBS)(25 μL/well). Wells were emptied and then blocked for two hours with 10%fetal bovine serum (FBS; Invitrogen, Carlsbad, Calif.) in PBS (PBS-10%FBS). Wells were washed 3× with PBS containing 0.05% tween-20 (PBS-T)and samples were introduced into the wells in a total volume of 100 μLconsisting of 25 μL human plasma and 75 μL PBS-5% FBS. ICAM-5 standardcurves were prepared similarly in buffer containing 25 μL equine plasma(human ICAM-5-free) (Invitrogen, Carlsbad, Calif.), to control for theeffects of sample matrix. Plates were incubated for three hours, washedand then incubated for one hour with biotinylated goat anti-human ICAM-5antibody purified by human ICAM-5 affinity chromatography (R&D Systems;catalog #BAF1950; 1 μg/ml in PBS-1% FBS; 25 μL/well). Plates were washedand reacted for one hour with MSD® SULFO-TAG labeled streptavidindetection reagent (Meso Scale Discovery; catalog# R32AD; 1 μg/ml in PBScontaining 1% bovine serum albumin (BSA); 25 μL/well). Plates werewashed, treated with the addition of MSD Read Buffer (Meso ScaleDiscovery; catalog# R92TC; 150 μL/well) and electrochemiluminescenceread using a SECTOR® Imager 6000 instrument (Meso Scale Discovery). Allincubations were carried out at room temperature with the exception ofthat for the capture antibody which was carried out at 4° C. The assaywas sensitive to less than 0.34 ng/ml as defined by theelectochemiluminescence signal value that was 10 times the standarddeviation above the mean electrochemiluminescence signal recorded forthe 0 ng ICAM-5 standard (N=10).

Detection/Quantification of BDNF

Levels of immunoreactive BDNF in plasma were measured in a mannersimilar to sICAM-5 (Example 1) using antibodies and BDNF standardprotein provided in the R&D Systems human BDNF ELISA Development Kit(catalog # DY248, Meso Scale Discovery). Detection is byelectrochemiluminescence using the MSD® SULFO-TAG labeled streptavidindetection reagent and SECTOR® Imager 6000 instrument (Meso ScaleDiscovery). The assay is sensitive to less than 0.08 ng/ml as defined bythe electochemiluminescence signal value that was 10 times the standarddeviation above the mean electrochemiluminescence signal recorded forthe 0 ng BDNF standard (N=10).

Detection/Quantification of Other Polypeptides

Two multiplexed assays for cytokines and chemokines were used foranalysis of patient and control plasma samples on the SECTOR® Imager6000 instrument (Meso Scale Discovery, Gaitherburg, Md.) The first ofthese assays is the Human ProInflammatory 9 Plex™ Assay for themeasurement of IL-2, IL-8, IL-12p70, IL-1β, GM-CSF, IFN-γ, IL-6, IL-10and TNF-α (MesoScale catalog #K15007C-4). The second of these assays isthe Human Chemokine 9 Plex™ Assay for the measurement of Eotaxin,MIP-1β, Eotaxin-3, TARC, IP-10, IL-8, MCP-1, MDC, and MCP-4 (catalog#K15001C-1). The samples are added to plates that were pre-coated withcapture antibodies for the specific cytokines. The plates was sealed andshaken at room temperature for two hours. The plates were washed inPBS+0.05% Tween-20 and detection antibody solution (1× or 1 μg/mL) isadded. The plates were once again sealed and set to shake at roomtemperature for two hours. The plates were then washed once more inPBS+0.05% Tween-20. Read buffer was added at a 2× concentration and theplate was read on the SECTOR® 6000 Imager.

Other assays prepared by one of skill in the art or commerciallyavailable are used for additional polypeptides.

Results

Human epilepsy patient samples had altered levels of one or morepolypeptides relative to control. Data is reflected in Table 1, below,and in FIGS. 2A-2C through 13A-13C. The data is described in the BRIEFDESCRIPTION OF THE DRAWINGS, above. Analyzed with a Receiver OperatingCharacteristic/Condition (ROC) calculation, the area under the curve(AUC), which is a measure of how well the assay detects epilepsy, was0.70 or greater where p≦0.05 (an AUC value of 1.0 would reflect aperfect diagnostic).

TABLE 1 Analyte Epilepsy¹ Epilepsy n Controls¹ Controls n Ratio²p-value³ AUC⁴ IL-1β 0.3 ± 0.1 12 0.1 ± 0.0 10 ↑4.0 0.003 0.80 sICAM54.24 ± 1.14 (ng/ml) 13 15.72 ± 3.63 (ng/ml)  16 ↓3.7 0.003 0.80 IL-6 3.1± 0.8 17 1.1 ± 0.2 26 ↑2.8 0.012 0.82 TARC 197 ± 62  12 77 ± 13 9 ↑2.60.035 0.76 IL-2 0.6 ± 0.1 16 0.3 ± 0.0 26 ↑2.4 0.008 0.79 IFN-γ 1.6 ±0.4 17 0.7 ± 0.1 24 ↑2.4 0.010 0.70 IL-10 2.9 ± 1.3 16 1.8 ± 0.3 26 ↑1.60.191 0.61 IL-12p70 1.1 ± 0.2 16 1.6 ± 0.3 26 ↓1.5 0.079 0.53 IL-8 4.2 ±0.4 24 2.9 ± 0.2 26 ↑1.4 0.002 0.72 TNF-α 7.3 ± 1.5 17 5.5 ± 0.4 26 ↑1.30.131 0.56 MCP-1 269 ± 33  12 219 ± 13  9 ↑1.2 0.082 0.73 MDC 2.51 ±0.28 (ng/ml) 12 2.17 ± 0.18 (ng/ml) 9 ↑1.2 0.145 0.70 MIP-1β 67.4 ± 9.6 12 58.4 ± 5.9  9 ↑1.2 0.207 0.60 GM-CSF 1.2 ± 0.3 17 1.4 ± 0.4 23 ↓1.20.297 0.55 MCP-4 414 ± 69  12 367 ± 67  9 ↑1.1 0.306 0.56 IP-10 187 ±34  12 207 ± 38  9 ↓1.1 0.346 0.57 BDNF 1.03 ± 0.16 (ng/ml) 15 0.96 ±0.20 (ng/ml) 10 ↑1.1 0.383 0.53 Eotaxin-3 6.5 ± 0.7 12 6.4 ± 1.1 9 ↑1.00.463 0.55 Eotaxin 538 ± 99  12 525 ± 105 9 ↑1.0 0.462 0.56 TARC/sICAM5(122.8 ± 53.9) × 10⁻³   10 (7.2 ± 2.5) × 10⁻³ 8 ↑17.1 0.025 1.00IL6/sICAM5 (1.9 ± 1.0) × 10⁻³ 13 (0.2 ± 0.1) × 10⁻³ 16 ↑9.9 0.050 0.90IL8/sICAM5 (3.3 ± 1.3) × 10⁻³ 18 (0.5 ± 0.2) × 10⁻³ 16 ↑6.5 0.017 0.88¹average ± sem, (pg/ml unless otherwise labeled). ²(↑) Increased inepilepsy (↓)Decreased in epilepsy. ³one-tailed t-test. ⁴Area under theCurve of the ROC curve.

Any document (including but not limited to any patent, patentapplication, publication, and website) listed herein is herebyincorporated herein by reference in its entirety. While thesedevelopments have been disclosed with reference to specific embodiments,it is apparent that other embodiments and variations of this inventionare devised by others skilled in the art without departing from the truespirit and scope of the developments. The appended claims include suchembodiments and variations thereof.

1. A method for predicting or detecting seizure in a mammalian subjectcomprising: contacting a blood plasma or blood serum sample obtainedfrom a mammalian subject with a diagnostic reagent that can measure ordetect the expression level of soluble ICAM-5 (sICAM-5); and contactingsaid blood plasma or blood serum sample obtained from a mammaliansubject with a diagnostic reagent that can measure or detect theexpression level of TARC; wherein a decreased plasma or serumconcentration of sICAM-5 in said blood plasma or blood serum sample ofsaid mammalian subject, in combination with an increased plasma or serumconcentration of TARC in said blood plasma or blood serum sample of saidmammalian subject, indicates a tendency to have seizure.